Abstract
Phenethyl isothiocyanate (PEITC) and sulforaphane (SF) exhibit tumor preventive activity in lung, prostate, breast and colon cancers. Our objective was to examine the effect of these two isothiocyanates on estrogen receptor-related genes, and genes related to apoptosis and cell cycle in the estrogen-dependent breast cancer cell line MCF7 and in normal human epithelial breast (HME) cells. We treated cells with 0.3 μM or 3.0 μM concentrations of PEITC or SF. In HME cells, gene expression was significantly altered for 23 genes by PEITC at a concentration of 0.3 μM and 4 genes at 3.0 μM. SF altered the expression of 16 genes at a concentration of 0.3 μM and 2 genes at 3.0 μM. In HME cells, genes altered by both PEITC and SF exhibited changes in gene expression that were similar in extent as well as direction of change. In MCF-7 cells, PEITC did not produce any significant changes in the gene expression at both treatment levels. SF produced significant changes in 7 genes, but only at the higher treatment level of 3.0 μM. Normal mammary cells exhibited more changes in the expression of estrogen receptor related genes than did breast cancer cells, and significantly these changes occurred predominantly at the low concentration of 0.3 μM, a concentration achievable by dietary input of isothiocyanates. Novel findings were the upregulation of the pro-apoptotic gene BAD and estrogen receptor beta gene in normal human mammary cells. These gene alterations observed, along with upregulation of tumor suppressors p21 and p27, may provide a protective effect to mammary cells against breast cancer.
Keywords: phenethyl isothiocyanate, sulforaphane, human mammary epithelial cells, breast cancer MCF-7 cells, breast cancer prevention, gene expression
Introduction
The inverse correlation between consumption of cruciferous vegetables and cancer risk has been demonstrated in lung, colon, breast, stomach and prostate cancers (1–3). Organic isothiocyanates, one of the components of cruciferous vegetables, have been identified as a class of compounds that may cause this effect. Isothiocyanates (general chemical structure R-N=C=S) occur in crucifers as glucosinolate precursors. Isothiocyanates inhibit the metabolic activation of a variety of carcinogens that occur in tobacco products and the diet (4). More recent studies have uncovered additional pathways such as induction of apoptosis which can explain the anti-carcinogenic actions of isothiocyanates (5). Sulforaphane (SF) and phenethyl isothiocyanate (PEITC) are two organ isothiocyanates obtained mainly from broccoli and watercress, respectively (6). An epidemiologic study with 720 breast cancer cases and 810 controls reported that the consumption of broccoli is inversely associated with breast cancer risk in pre-menopausal women (1). In vitro, PEITC is cytotoxic in human breast cancer MCF-7 and human mammary epithelial MCF-12A cells, with IC50 values of 7.32 ± 0.25 μM and 7.71 ± 0.07 μM, respectively (7). A maximal plasma concentration of approximately 1.0 μM is achieved by consumption of 100 grams of watercress (8). In order to investigate the mechanisms by which these ITCs can exert preventive and cytotoxic effects in breast cancer, we used a human breast cancer gene array with 96 genes (SuperArray Inc.) to assess changes in gene expression.
Most in vitro experiments have so far focused on the effect of high treatment levels of ITCs on cancer cells. However, the most likely exposure of isothiocyanates is exposure to low levels in human beings who do not have breast cancer. The effect of isothiocyanates on mammary cells in such an environment, to our knowledge, has not been studied. Our objective was to study the effect of two common dietary ITCs, PEITC and SF, on gene expression changes in normal and cancerous human mammary epithelial cells, and to determine chemopreventive potential of isothiocyanates in breast cancer. Our hypothesis is that the changes in gene expression between normal and breast cancer cell lines are distinct and these changes may be important when evaluating the “protective” effects of isothiocyanates. Changes in gene expression may be able to explain the apparent protective effects of isothiocyanates against breast cancer.
Materials and Methods
Materials
Mammary Epithelial Basal Medium, epidermal growth factor, hydrocortisone, insulin, and bovine pituitary extract were purchased from Cambrex Corp. (these items are now available from Lonza Inc., Walkersville, MD). Transferrin, isoproterenol and dimethyl sulfoxide were obtained from Sigma Aldrich. PEITC and SF were obtained from LKT Laboratories (St. Paul, MN). RPMI1640, penicillin, streptomycin, fetal bovine serum and MMLV reverse transcriptase were obtained from Invitrogen (Grand Island, NY). GEArray Q series Estrogen Receptor Signaling Gene Arrays and Ampolabeling LPR (linear polymerase reaction) kit were obtained from SABiosciences Inc. (Frederick, MD). SV RNA isolation kit was obtained from Promega Corporation (Madison, WI). MCF7 cells were provided by Dr. Susan E. Bates (National Cancer Institute, Bethesda, MD) and HME cells were provided by Dr. Martha Stampfer (Lawrence Berkeley National Laboratory, Berkeley, CA). The HME cells represent normal finite lifespan mammary cells which were obtained from reduction mammoplasty tissue of a 21-year-old woman.
Methods
Cell Culture
HME cells were incubated in 75 ml flasks until 60–80% confluence in Mammary Epithelial Basal Medium supplemented with 5 ng/ml epidermal growth factor, 500 ng/ml hydrocortisone, 5 μg/ ml insulin, 70 μg/ml bovine pituitary extract, 5 μg/ml transferrin, and 10−5 M isoproterenol at 1% CO2 in a 37°C incubator. The cells were treated with dimethyl sulfoxide 0.015% v/v (control), SF or PEITC at one of two concentrations (0.3 μM or 3.0 μM) for 48 hours (n = 3). These concentrations are similar to the plasma levels of PEITC that can be achieved after ingestion of 100 gm of watercress (8). MCF-7 cells in 75 ml flasks were incubated in RPMI1640 supplemented with 100 units/ml penicillin, and 100 μg/ml of streptomycin and 10% fetal bovine serum until 60–80% confluence at 5% carbon dioxide in a 37°C incubator. Cells were treated with dimethyl sulfoxide 0.015% v/v (control), SF or PEITC at one of two concentrations (0.3 or 3.0 μM) for 48 hours. At the end of the incubation period, all cells except HME cells at 3.0 μM were harvested using ice-cold phosphate buffer saline and cell scraping. HME cells at 3.0 μM were harvested via direct application of lysis buffer from the RNA isolation kit.
RNA Isolation and cDNA Formation
Total RNA was isolated from both cell lines by using the SV RNA Isolation System and quantified spectrophotometrically at 260 nm. cDNA was prepared from total RNA by reverse transcription with MMLV reverse transcriptase or using ampolabeling LPR kit.
Hybridization and Imaging
GEArray Q series Estrogen Receptor Signaling Gene Arrays were employed according to the manufacturer’s instructions. Ninety-six genes were used to study the expression profile of the genes involved in estrogen receptor signaling pathways. cDNA was chemiluminescence-labeled using biotin, hybridized under precisely specified conditions to a positively charged nylon membrane containing the arrayed DNA. After washing, the relative expression level of each gene was analyzed using a Kodak Image Station 440CF.
Normalization and Statistical Analysis
The mean intensity for each gene spot was measured. The mean intensity of the background PUC18/Blank genes was subtracted from mean intensity to give net mean intensity. The average of total intensity on the array was used to normalize the intensity of the gene spots. The average intensity for a gene for each treatment was compared with the control group, with the Student’s t test, with the level of significance at 0.05. Significance Analysis of Microarrays (SAM) was also used to analyze the data, which accounts for errors arising from repeated measurements (9). While using SAM, delta was set such that the false discovery rate for each array was minimized. The false detection rate for comparisons ranged from 0–1%. Results from both tests were compared, and genes that were significant by both tests are reported.
Results
As shown in Table 1, in normal as well as cancer cells, isothiocyanates produced significant gene expression changes in a number of genes. In HME cells, gene expressions were significantly altered for 23 genes by PEITC at a concentration of 0.3 μM and 4 genes at a concentration of 3.0 μM. SF altered the expression of 16 genes at 0.3 μM and 2 genes at 3.0 μM. In HME cells, genes altered by both PEITC and SF exhibited changes in gene expression that were similar in extent (fold change) as well as direction of change (up- or downregulation). In MCF-7 cells, PEITC did not produce any significant changes in the gene expression at both treatment levels. SF produced a significant change in 7 genes, only at the higher treatment level of 3.0 μM in MCF-7 cells. Isothiocyanates altered the expression of more genes in human mammary epithelial cells than breast cancer cells. Genes altered were related to (i) apoptosis and cell cycle regulation (example: BAD, p21, p27), (ii) cell adhesion (example: claudin-7, fibronectin), (iii) estrogen receptor signaling (example: estrogen receptor beta) and (iv) prognostic cancer markers (example: her2, EGFR). Genes significantly affected by treatment are listed in Table 2. A listing of the genes present in the GEArray Q Series Estrogen Receptor Signaling Gene Array can be found in the Appendix.
Table 2.
Effect of Isothiocyanates on Gene Expression in Human Mammary Epithelial (HME) and Human Breast Cancer MCF-7 Cells
| GeneBank number | Gene description | Fold change |
|---|---|---|
| 0.3 μM PEITC on HME cells | ||
| NM_003248 | Thrombospondin 4a | Inf |
| NM_013280 | Fibronectin leucine rich transmembrane protein 1 | 147.3 |
| NM_002581 | Pregnancy-associated plasma protein A | 38.42 |
| NM_005954 | Metallothionein 3 (growth inhibitory factor (neurotrophic)) | 30.52 |
| NM_004322 | BCL2-antagonist of cell death | 24.08 |
| NM_005438 | FOS-like antigen 1 | 22.95 |
| NM_001237 | Cyclin A2 | 22.36 |
| NM_182741 | Mucin 1, transmembrane | 21.88 |
| NM_002228 | V-jun sarcoma virus 17 oncogene homolog (avian) | 21.20 |
| NM_000044 | Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease) | 19.86 |
| NM_004448 | V-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) | 17.94 |
| NM_004064 | Cyclin-dependent kinase inhibitor 1B (p27, Kip1) | 14.79 |
| NM_002417 | Antigen identified by monoclonal antibody Ki-67 | 13.01 |
| NM_002407 | Secretoglobin, family 2A, member 1 | 11.94 |
| NM_014330 | Protein phosphatase 1, regulatory (inhibitor) subunit 15A (GADD34) | 7.923 |
| NM_005694 | COX17 homolog, cytochrome c oxidase assembly protein (yeast) | 7.829 |
| NM_001437 | Estrogen receptor 2 (ERbeta) | 7.555 |
| NM_003486 | Solute carrier family 7 (cationic amino acid transporter, y+ system), member 5 CD98 | 5.594 |
| NM_000389 | Cyclin-dependent kinase inhibitor 1A P21/Waf1/CIP1 | 5.281 |
| NM_002284 | Keratin, hair, basic, 6 (monilethrix) | 4.344 |
| NM_000213 | Integrin, beta 4 | 3.981 |
| NM_001993 | Coagulation factor III (thromboplastin, tissue factor) | −2.355 |
| NM_001909 | Cathepsin D (lysosomal aspartyl protease) | −8.045 |
| NM_001307 | Claudin 7 | −26.04 |
| 3.0 μM PEITC on HME cells | ||
| NM_001993 | Coagulation factor III (thromboplastin, tissue factor) | −3.937 |
| NM_003236 | Transforming growth factor, alpha | −3.279 |
| NM_021130 | Homo sapiens peptidylprolyl isomerase A (cyclophilin A) (PPIA) | −5.102 |
| NM_005978 | S100 calcium binding protein A2 (CaN19) | −2.096 |
| 0.3 μM SF on HME cells | ||
| NM_000600 | Interleukin 6 (interferon, beta 2) | 61.51 |
| NM_005954 | Metallothionein 3 (growth inhibitory factor (neurotrophic)) | 37.98 |
| NM_002581 | Pregnancy-associated plasma protein A | 34.34 |
| NM_182741 | Mucin 1, transmembrane | 22.96 |
| NM_002228 | V-jun sarcoma virus 17 oncogene homolog (avian) | 22.06 |
| NM_000044 | Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease) | 19.88 |
| NM_004448 | V-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) | 16.76 |
| NM_003247 | Thrombospondin 2 | 15.20 |
| NM_002407 | Secretoglobin, family 2A, member 1 | 9.470 |
| NM_003226 | Trefoil factor 3 (intestinal) | 9.378 |
| NM_001437 | Estrogen receptor 2 (ERbeta) | 7.882 |
| NM_005694 | COX17 homolog, cytochrome c oxidase assembly protein (yeast) | 7.078 |
| NM_000389 | Cyclin-Dependent Kinase Inhibitor 1A | 6.606 |
| NM_002284 | Keratin, hair, basic, 6 (monilethrix) | 5.224 |
| NM_000610 | CD44 antigen (homing function and Indian blood group system) | 2.391 |
| NM_005228 | Epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) | 2.380 |
| 3.0 μM SF on HME cells | ||
| NM_001993 | Coagulation factor III (thromboplastin, tissue factor) | −4.950 |
| NM_001909 | Cathepsin D (lysosomal aspartyl protease) | −1.923 |
| 3.0 μM SF on MCF7 cells | ||
| NM_000077 | Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) | 19.13 |
| NM_003246 | Thrombospondin 1 | 10.11 |
| NM_000610 | CD44 antigen (homing function and Indian blood group system) | 9.601 |
| NM_000988 | Ribosomal protein L27 | 2.870 |
| NM_021130 | Homo sapiens peptidylprolyl isomerase A (cyclophilin A) (PPIA) | 1.642 |
| NM_003486 | Solute carrier family 7 (cationic amino acid transporter, y+ system), member 5 | −6.11 |
| NM_002051 | GATA binding protein 3 | −13.70 |
Thrombospondin 4 did not have any signal in the controls, while it showed signal in the treated group, leading to values of “infinity” in fold
Discussion
Isothiocyanates are compounds derived from cruciferous vegetables such as broccoli, cabbage and watercress. Based on epidemiological studies, isothiocyanates are widely recommended as cancer preventive agents and commercially available in herbal supplements (1, 10, 11). The purpose of this study was to evaluate these isothiocya-nates as breast cancer preventive agents. Breast cancer progression is determined by changes in cellular proliferation, apoptosis and metastasis. The estrogen receptor signaling pathway in estrogen receptor positive breast epithelial cells provides an additional mechanism for cellular proliferation (12). We examined the effects of PEITC and SF on expression of genes related to estrogen receptor signaling and other pathways important in the development of breast cancer using primary cultures of normal human mammary epithelial cells and the cancerous estrogen receptor-positive breast cancer cell line MCF7. Comparing the effects of ITCs on normal versus cancer cell lines will provide insight in their role in the prevention of breast cancer.
Genes Related to Apoptosis and Cellular Proliferation
Mechanisms of cancer prevention include the induction of apoptosis and reduction of cellular proliferation. Isothiocyanates have been reported to induce apoptosis and reduce cellular proliferation in a number of cancer cell lines in vitro (13, 14). We report for the first time induction of BAD, a pro-apoptotic gene by 0.3 μM PEITC in mammary cells. A time-dependent upregulation of BAD, along with increase in apoptosis has been observed in pre-adipocyte cell line AML-I treated with 100 μmol/L quercetin, a dietary flavonoid (15). BAD belongs to the Bcl-2 family of proteins and regulates apoptosis (16, 17). BAD is downregulated completely by estradiol in MCF7s cells (18). Isothiocyanates have been shown to upregulate the related protein BAX in vitro as well as in vivo (19, 20). Other genes reported to have pro-apoptotic effects including THSB4 and GADD34 were also upregulated by PEITC. GADD34 is reported as a pro-apoptotic growth suppressor. Other GADD family members have been reported to be induced by 25 μM PEITC treatment in human adenocarci-noma HCT-116 cells (21).
Anti-apoptotic and proliferative effects observed include upregulation of v-jun and Ki67. V-jun, a known oncogene, was upregulated by both PEITC and SF. ITC-induced activation of AP-1 pathway (of which v-jun is a member) has previously been reported. Concentrations of 5–10 μM PEITC and SF produce significant activation of AP-1 activity as measured by luciferase activity assay when incubated with the prostate cancer cell line PC3-C9 and the bladder cancer cell line UM-UC-3 (22, 23). Ki67, a cell proliferation marker associated with apoptosis, was upregu-lated by both PEITC and SF at 0.3 μM concentrations.
Metallothionein 3 (MT3) was significantly upregulated by PEITC. While overexpression of this gene has been shown to inhibit growth of certain breast cancer cell lines including MCF7, expression of MT3 has also been correlated to higher grade tumors and poor cancer prognosis (24, 25).
To summarize, isothiocyanates induced cyclin-dependent kinase inhibitors, but other effects, including the induction of Ki67 and v-Jun, make it difficult to determine if induction of cyclin-dependent kinase inhibitors will result in apoptosis. In HME cells, SF induces anti-apoptosis, but no pro-apoptosis or proliferation genes; therefore, PEITC may have greater effects than SF on the induction of apoptosis in mammary cells.
Genes Related to Cell Adhesion
Cell adhesion is an important feature of cellular scaffolding, which prevents cells from breaking away from the existing tissue and migrating to another location. A decrease in the adhesion molecules such as E-cadherin has been shown to be correlated with increased metastasis, while fibronectin can increase cellular motility of breast cancer cells (26, 27). In the present study, we found that a number of cell adhesion molecules were altered by isothiocyanate treatment. Following treatment with 0.3 μM PEITC or SF, both altered motility facilitating cell adhesion molecules (fibronectin (FLRT1), claudin-7, integrin-b4 and episialin by PEITC and CD44 by SF). Claudin-7 expression is reported to be lower in invasive carcinomas than in normal breast tissue, and knockdown of claudin-7 expression leads to loss of E-cadherin and increased invasiveness in squamous cell carcinoma cells. Promotion of cell motility and enhancement of metastatic features may signal a harmful effect of isothiocyanates. The role of isothiocyanates in the loss of cell adhesion has previously been reported; 12 μM allyl isothiocyanate has been shown to increase cell detachment in HT-29 colorectal cells (28, 29).
Estrogen Receptor and Gene Interactions
Estrogen receptors alpha and beta play an important role in the regulation of cell growth and proliferation in mammary cells. Several studies have shown that the actions of these two receptors are often opposing. ERalpha may enhance cellular proliferation of MCF7 cells while the addition or endogenous expression of ERbeta by these cells reduces their proliferation by causing cell cycle arrest in G2 phase (30). We saw an increase in ERbeta gene expression in HME cells following treatment with 0.3 μM of PEITC and SF. This was accompanied by increased expression of p21 and p27 mRNA. A less than 5-fold increase on p21 and p27 protein was observed on treatment of PC-3 cells with indole-3-carbinol for 48 hours (31). Comparable treatments of 30 μM showed significant reductions in cell proliferation under the same conditions. The effect of ERbeta on gene expression of these two tumor suppressor genes has been shown previously in MCF7 cells (30). Cyclin A and Ki67, which were upregulated by 0.3 μM PEITC in our study, have been shown to be elevated by ERbeta expression (32). Cyclin A peaks in the G2 phase, further indicating that ITCs, especially PEITC, may be causing G2/M phase arrest in HME cells at low treatment levels. G2 arrest through the upregulation of genes such as ERbeta, p21 and p27 may represent a mechanism of cancer prevention of PEITC.
Prognostic Cancer Markers
The upregulation of Her2 by PEITC and co-upregulation of her2 and EGFR by SF in normal breast cells may be a matter of concern when evaluating ITC effects. These effects may represent a “pro-cancerous” effect of ITCs if not overshadowed by their other beneficial effects, such as p21 upregulation. More studies of effects of ITCs on cell cycle and her2 in breast cells are necessary to determine their effects in breast cancer prevention.
Gene Expression Changes Dependent on Concentration and Cell Type
MCF7 cells appear to be more resistant to gene expression changes than HME cells, which may mean that some of the regulatory mechanisms responsible for these changes may be transformed in the conversion of normal to mammary cancer cells.
IC50s of SF and PEITC in MCF7 cells are known and the concentrations of treatment are much lower than IC50s of these compounds. (7) However, IC50s in HME cells have not been investigated. It is possible that the ITCs are more cytotoxic to HME cells and therefore produce more changes in apoptosis and cell proliferation-related cells. However, this needs to be investigated.
Additionally, it is observed that lower concentrations of isothiocyanates exhibit a different gene expression pattern than the higher concentrations; metabolism may contribute to the changes observed. Part of the changes that we see may be caused by active metabolites of isothiocyanates. Induction of apoptosis in human alveolar basal epithelial cells A549 by phenethyl isothiocyanate-N acetylcysteine has been demonstrated (33). The ratios of metabolites to parent may change depending on starting concentrations of the isothiocyanates, and this may lead to different genes being affected. Assessment of metabolism of ITCs in HME cells may provide explanations for the apparent differences in expression.
Conclusion
Overall, isothiocyanates have numerous effects on gene expression in human mammary cells. A very significant finding was the greater number of changes in gene expression observed at dietary concentrations of isothiocyanates (0.3 μM) compared with that observed following a 10-fold higher concentration. Several effects observed in HME cells are consistent with reports of ITCs effects in other cancer cell lines. Novel findings were the upregulation of the pro-apoptotic gene BAD and estrogen receptor beta gene in normal human mammary epithelial cells. These gene alterations observed, along with upregu-lation of tumor suppressors p21 and p27, may provide a protective effect to mammary cells against breast cancer. However, we need to be cautious about the net effects of isothiocyanates as several other alterations, such as upregulation of her2 and EGFR, may present unfavorable effects of isothiocyanates. These need to be investigated in order to further understand the effects of isothiocyanates on breast cancer. Additional studies such as determining contribution of metabolites and parent compounds to apoptosis and cellular adhesion in normal versus cancer cell lines up on treatment of cells at these concentrations will be helpful in determining the final effects of these treatments on the cells.
Table 1.
Genes Altered by Isothiocyanate Treatment in Mammary Cells
| Treatment level | HME cells | MCF7 cells |
|---|---|---|
| 0.3 μM PEITC | 23 | - |
| 3.0 μM PEITC | 4 | - |
| 0.3 μM sulforaphane | 16 | - |
| 3.0 μM sulforaphane | 2 | 7 |
Acknowledgments
Support was provided by NIH grant CA121404. UT was supported in part by a fellowship from Daiichi Sankyo Pharmaceuticals Inc.
We thank Elizabeth A. Scott-Ramsay for her contributions to this project.
Appendix.
Layout of GEArray Q Series Human Breast Cancer and Estrogen Receptor Signaling Gene Array (from SuperArray, Inc.)
| Position | GeneBank | Symbol | Description | Gene name |
|---|---|---|---|---|
| 1 | NM_000044 | AR | Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease) | AR |
| 2 | NM_001185 | AZGP1 | Alpha-2-glycoprotein 1, zinc | AZGP1 |
| 3 | NM_004322 | BAD | BCL2-antagonist of cell death | Bad |
| 4 | NM_004323 | BAG1 | BCL2-associated athanogene | BAG-1 |
| 5 | NM_000633 | BCL2 | B-cell CLL/lymphoma 2 | Bcl-2 |
| 6 | NM_004050 | BCL2L2 | BCL2-like 2 | Bcl-w |
| 7 | NM_015548 | BPAG1 | Bullous pemphigoid antigen 1, 230/240kDa | BPAG1 |
| 8 | NM_000064 | C3 | Complement component 3 | C3 |
| 9 | NM_003914 | CCNA1 | Cyclin A1 | Cyclin A1 |
| 10 | NM_001237 | CCNA2 | Cyclin A2 | Cyclin A |
| 11 | NM_053056 | CCND1 | Cyclin D1 (PRAD1: parathyroid adenomatosis 1) | Cyclin D1 |
| 12 | NM_001238 | CCNE1 | Cyclin E1 | Cyclin E1 |
| 13 | NM_004702 | CCNE2 | Cyclin E2 | Cyclin E2 |
| 14 | NM_000610 | CD44 | CD44 antigen (homing function and Indian blood group system) | CD44 |
| 15 | NM_004360 | CDH1 | Cadherin 1, type 1, E-cadherin (epithelial) | E-cadherin |
| 16 | NM_000389 | CDKN1A | Cyclin-dependent kinase inhibitor 1A | P21/Waf1/CIP1 |
| 17 | NM_004064 | CDKN1B | Cyclin-dependent kinase inhibitor 1B (p27, Kip1) | p27Kip1 |
| 18 | NM_000077 | CDKN2A | Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) | p16INK4 |
| 19 | NM_001307 | CLDN7 | Claudin 7 | CLDN7 |
| 20 | NM_001831 | CLU | Clusterin (complement lysis inhibitor, SP-40,40, sulfated glycoprotein 2, testosterone-repressed prostate message 2, apolipoprotein J) | TRPM2/SP-40/ APOJ |
| 21 | NM_001848 | COL6A1 | Collagen, type VI, alpha 1 | COL6A1 |
| 22 | NM_005694 | COX17 | COX17 homolog, cytochrome c oxidase assembly protein (yeast) | COX17 |
| 23 | NM_001904 | CTNNB1 | Catenin beta 1 | b Catenin |
| 24 | NM_001908 | CTSB | Cathepsin B | Cathepsin B |
| 25 | NM_001909 | CTSD | Cathepsin D (lysosomal aspartyl protease) | Cathepsin D |
| 26 | NM_000103 | CYP19A1 | Cytochrome P450, family 19, subfamily A, polypeptide 1 | ARO1 |
| 27 | NM_006094 | DLC1 | Deleted in liver cancer 1 | DLC1 |
| 28 | NM_005228 | EGFR | Epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) | EGFR |
| 29 | NM_004448 | ERBB2 | V-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) | TKR1/Her-2 |
| 30 | NM_000125 | ESR1 | Estrogen receptor 1 | ER alpha |
| 31 | NM_001437 | ESR2 | Estrogen receptor 2 (ERbeta) | ER-beta-cx |
| 32 | NM_001993 | F3 | Coagulation factor III (thromboplastin, tissue factor) | TF |
| 33 | NM_000800 | FGF1 | Fibroblast growth factor 1 (acidic) | FGF1 |
| 34 | NM_013280 | FLRT1 | Fibronectin leucine rich transmembrane protein 1 | FLRT1 |
| 35 | NM_005438 | FOSL1 | FOS-like antigen 1 | Fra-1 |
| 36 | NM_014211 | GABRP | Gamma-aminobutyric acid (GABA) A receptor, pi | GABRP |
| 37 | NM_002051 | GATA3 | GATA binding protein 3 | GATA3 |
| 38 | NM_080425 | GNAS | GNAS complex locus | GNAS1 |
| 39 | NM_000177 | GSN | Gelsolin (amyloidosis, Finnish type) | Gelsolin |
| 40 | NM_002128 | HMGB1 | High-mobility group box 1 | HMG1 |
| 41 | NM_001540 | HSPB1 | Heat shock 27kDa protein 1 | HSP28/HSP27/ Hsp25 |
| 42 | NM_002166 | ID2 | Inhibitor of DNA binding 2, dominant negative helix-loop-helix protein | ID2 |
| 43 | NM_000597 | IGFBP2 | Insulin-like growth factor binding protein 2, 36kDa | IGFBP-2 |
| 44 | NM_000417 | IL2RA | Interleukin 2 receptor, alpha | CD25 |
| 45 | NM_000600 | IL6 | Interleukin 6 (interferon, beta 2) | IL-6 |
| 46 | NM_000565 | IL6R | Interleukin 6 receptor | IL-6 Ra |
| 47 | NM_002184 | IL6ST | Interleukin 6 signal transducer (gp130, oncostatin M receptor) | GP130 |
| 48 | NM_000210 | ITGA6 | Integrin, alpha 6 | Integrin a6 |
| 49 | NM_000213 | ITGB4 | Integrin, beta 4 | Integrin b4 |
| 50 | NM_002228 | JUN | V-jun sarcoma virus 17 oncogene homolog (avian) | V-jun |
| 51 | NM_000222 | KIT | V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog | KIT |
| 52 | NM_001730 | KLF5 | Kruppel-like factor 5 (intestinal) | GC Box BP |
| 53 | NM_012427 | KLK5 | Kallikrein 5 | KLK5 |
| 54 | NM_000224 | KRT18 | Keratin 18 | KRT18 |
| 55 | NM_002276 | KRT19 | Keratin 19 | Keratin 19 |
| 56 | NM_002284 | KRTHB6 | Keratin, hair, basic, 6 (monilethrix) | KRTHB6 |
| 57 | NM_023009 | MLP | MARCKS-like protein | MacMarcks |
| 58 | NM_005043 | MAP2K7 | Mitogen-activated protein kinase kinase 7 | JNKK2/MKK7 |
| 59 | NM_002417 | MKI67 | Antigen identified by monoclonal antibody Ki-67 | Ki67 (MKI67) |
| 60 | NM_005954 | MT3 | Metallothionein 3 (growth inhibitory factor (neurotrophic)) | MT3 |
| 61 | NM_182741 | MUC1 | Mucin 1, transmembrane | Episialin |
| 62 | NM_006166 | NFYB | Nuclear transcription factor Y, beta | NFYB |
| 63 | NM_002506 | NGFB | Nerve growth factor, beta polypeptide | NGF |
| 64 | NM_002507 | NGFR | Nerve growth factor receptor (TNFR superfamily, member 16) | NGFR |
| 65 | NM_000269 | NME1 | Non-metastatic cells 1, protein (NM23A) expressed in | NM23 |
| 66 | NM_002581 | PAPPA | Pregnancy-associated plasma protein A | PAPPA |
| 67 | NM_000926 | PGR | Progesterone receptor | PR |
| 68 | NM_002658 | PLAU | Plasminogen activator, urokinase | uPA |
| 69 | NM_014330 | PPP1R15A | Protein phosphatase 1, regulatory (inhibitor) subunit 15A | GADD34 |
| 70 | NM_000314 | PTEN | Phosphatase and tensin homolog (mutated in multiple advanced cancers 1) | PTEN |
| 71 | NM_000963 | PTGS2 | Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) | Cox-2 |
| 72 | NM_002872 | RAC2 | Ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2) | Rac2 |
| 73 | NM_000988 | RPL27 | Ribosomal protein L27 | RPL27 |
| 74 | NM_005978 | S100A2 | S100 calcium binding protein A2 | CaN19 |
| 75 | NM_006551 | SCGB1D2 | Secretoglobin, family 1D, member 2 | Lipophilin B |
| 76 | NM_002407 | SCGB2A1 | Secretoglobin, family 2A, member 1 | C3/Lipophilin |
| 77 | NM_002411 | SCGB2A2 | Secretoglobin, family 2A, member 2 | SCGB2A2 |
| 78 | NM_001085 | SERPINA3 | Serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 | AACT |
| 79 | NM_002639 | SERPINB5 | Serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 5 | Maspin |
| 80 | NM_000602 | SERPINE1 | Serine (or cysteine) proteinase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 | PAI-1 |
| 81 | NM_003486 | SLC7A5 | Solute carrier family 7 (cationic amino acid transporter, y+ system), member 5 | CD98 |
| 82 | NM_003125 | SPRR1B | Small proline-rich protein 1B (cornifin) | SPRR1B |
| 83 | NM_003714 | STC2 | Stanniocalcin 2 | STC2 |
| 84 | NM_003225 | TFF1 | Trefoil factor 1 (breast cancer, estrogen-inducible sequence expressed in) | pS2 |
| 85 | NM_003226 | TFF3 | Trefoil factor 3 (intestinal) | TFF3 |
| 86 | NM_003236 | TGFA | Transforming growth factor, alpha | TGF-a |
| 87 | NM_003246 | THBS1 | Thrombospondin 1 | TSP1 |
| 88 | NM_003247 | THBS2 | Thrombospondin 2 | Thrombospondin2 |
| 89 | NM_003248 | THBS4 | Thrombospondin 4 | THBS4 |
| 90 | NM_005424 | TIE | Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains | Tie-1 |
| 91 | NM_006291 | TNFAIP2 | Tumor necrosis factor, alpha-induced protein 2 | B94 |
| 92 | NM_000043 | TNFRSF6 | Tumor necrosis factor receptor superfamily, member 6 | Fas/Apo-1/CD95 |
| 93 | NM_000639 | TNFSF6 | Tumor necrosis factor (ligand) superfamily, member 6 | Fas ligand |
| 94 | NM_001067 | TOP2A | Topoisomerase (DNA) II alpha 170kDa | TOP2 alpha |
| 95 | NM_000546 | TP53 | Tumor protein p53 (Li-Fraumeni syndrome) | p53 |
| 96 | NM_003376 | VEGF | Vascular endothelial growth factor | VEGF |
| 97 | L08752 | PUC18 | PUC18 Plasmid DNA | pUC18 |
| 98 | L08752 | PUC18 | PUC18 Plasmid DNA | pUC18 |
| 99 | L08752 | PUC18 | PUC18 Plasmid DNA | pUC18 |
| 100 | — | Blank | — | — |
| 101 | — | Blank | — | — |
| 102 | — | Blank | — | — |
| 103 | NM_002046 | GAPD | Glyceraldehyde-3-phosphate dehydrogenase | GAPDH |
| 104 | NM_002046 | GAPD | Glyceraldehyde-3-phosphate dehydrogenase | GAPDH |
| 105 | NM_021130 | PPIA | Homo sapiens peptidylprolyl isomerase A (cyclophilin A) (PPIA) | Cyclophilin A |
| 106 | NM_021130 | PPIA | Homo sapiens peptidylprolyl isomerase A (cyclophilin A) (PPIA) | Cyclophilin A |
| 107 | NM_021130 | PPIA | Homo sapiens peptidylprolyl isomerase A (cyclophilin A) (PPIA) | Cyclophilin A |
| 108 | NM_021130 | PPIA | Homo sapiens peptidylprolyl isomerase A (cyclophilin A) (PPIA) | Cyclophilin A |
| 109 | NM_012423 | RPL13A | Ribosomal protein L13a | RPL13A |
| 110 | NM_012423 | RPL13A | Ribosomal protein L13a | RPL13A |
| 111 | NM_001101 | ACTB | Actin, beta | b-actin |
| 112 | NM_001101 | ACTB | Actin, beta | b-actin |
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